Commercial tissue microarray (Alenabio Biotechnology, Xi'an, China), composed of 8 samples of normal or cancer-adjacent breast tissues and 40 samples of breast cancer tissues, was used to evaluate the expression of TLR4 and NF-κB p65. The tumor tissues included 9 cases of Stage I disease, 25 cases of Stage II disease and 6 cases of Stage III disease.
Immunohistochemistry was performed on the human breast tissue microarray. The slides were deparaffinized and rehydrated in graded ethanol solutions. After wet autoclave pretreatment for antigen retrieval and suppressing endogenous peroxidase activity, the slides were blocked with 5% bovine serum albumin (BSA). The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used. Immunoreactivity was visualized using 3,3'-diaminobenzidine (Boster, Wuhan, China). Immunohistochemistry evaluation was performed independently by two researchers. A semi-quantitative HistoScore (H-score, ranged from 0 to 300) for each specimen was calculated by multiplying the percentage of positive areas (0–100%) by intensity (0 = nil; 1 = weak; 2 = moderate; 3 = strong) (Rojo et al., 2016 (link)).
Immunohistochemistry was performed on the human breast tissue microarray. The slides were deparaffinized and rehydrated in graded ethanol solutions. After wet autoclave pretreatment for antigen retrieval and suppressing endogenous peroxidase activity, the slides were blocked with 5% bovine serum albumin (BSA). The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used. Immunoreactivity was visualized using 3,3'-diaminobenzidine (Boster, Wuhan, China). Immunohistochemistry evaluation was performed independently by two researchers. A semi-quantitative HistoScore (H-score, ranged from 0 to 300) for each specimen was calculated by multiplying the percentage of positive areas (0–100%) by intensity (0 = nil; 1 = weak; 2 = moderate; 3 = strong) (Rojo et al., 2016 (link)).
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